Management of overwintering pine sawyer beetle, Monochamus alternatus with colonized Beauveria bassiana ERL836

Monochamus alternatus is a major forest pest that spreads pine wilt disease in pine trees as a vector of pine wilt nematodes. Chemical insecticides used as fumigants to control overwintering M. alternatus in forests are highly toxic to the environment, so we investigated entomopathogenic fungus Beauveria bassiana ERL836 as an eco-friendly and alternative material to control overwintering M. alternatus. In this work, we evaluated the insecticidal activity of B. bassiana ERL836 against M. alternatus adults, the possibility of fungal colonization on pine tree bark, and finally the control efficacy of fungal pre-treatment on pine tree logs against emerging M. alternatus adults in semi-field and field conditions. M. alternatus adults were killed on the pine tree logs pre-treated with the B. bassiana ERL836. White conidia were observed not only on the surface of the dead adults but also on the pine tree logs, suggesting that the adults were killed by the fungus on the pine. A formulated ERL836 powder treatment on larvae-infested pine logs showed high insecticidal activity against adults, similar to that with the fungal powder suspension treatment, but we demonstrated that using the fungal powder was simpler than using the suspension in field conditions. Even in the field condition, the fungal powder treatment showed high insecticidal activity against M. alternatus adults, which we attribute to its ability to maintain fungal activity for a long time in field conditions by covering the pine tree logs with a film during overwintering. We confirmed that the risk that fungus-infected M. alternatus adults would spread the fungus to other non-target forest insects was low. Thus, even a high-concentration treatment in a specific area is unlikely to transmit the fungus outside that area, so it can be safely used to control this pine wilt nematode vector in forest ecosystems.


Introduction
Pine wilt disease (PWD) is a serious problem in pine forests. The disease infects and damages pine trees, destroying forest ecosystems and lowering the quality of pine tree products, which dextrose agar medium (1/4 SDA, BD Difco TM , Sparks, MD, USA) in Petri dishes (dia. 60 mm) at 27˚C for 14 days to induce conidial production. The conidia of the strain were harvested by putting the agar blocks in which the strain was cultured in a 15-ml conical tube (SPL Life Science Co., Pocheon, Korea) with 5 ml of 0.03% siloxane solution (Silwet, FarmHannong Inc., Nonsan, Korea) and creating a suspension using a vortex mixer (WiseMix VM-10, Daihan, Korea). The numbers of conidia were counted three times at 400× magnification using a hemocytometer. The conidia suspension was diluted to 1.0 × 10 7 conidia/ml with 0.03% siloxane solution. Then, 2 μg of the conidia suspension was inoculated in 1/4 SDA medium at 27˚C for 24 h, and a conidia germination rate of >90% was confirmed. The experimental insects (Monochamus alternatus, Bombyx mori, Allomyrina dichotoma, and Lucanus maculifemoratus) were supplied by OsangKinsect (Yesan, Korea), an insect rearing company in Korea. All the insects were stored in insect breeding dishes at 25 ± 2˚C. The 4 th instars of B. mori larvae were used, and the adults of the others insects were used within ten days of emergence. Additionally, for the indoor and semi-field experiments with wintering pine trees, holders were drilled into a pine tree, and 5 th instar M. alternatus larvae were artificially inserted.

Insecticidal efficacy test of ERL836 against adults on pine tree in pot condition
The insecticidal activity of B. bassiana ERL836 against M. alternatus adults was evaluated by pre-treating pine trees (Pinus densiflora) with a fungal conidia suspension (1.0 ×10 6 or 1.0 ×10 7 conidia/ml) in pot conditions and then adding M. alternatus adults to the trees. Four pine trees with a height 80 cm were planted in a plastic pot (40 × 30 × 30 cm) with soil, and adequate water was supplied. The pine trees in pots were maintained for 7 days at 25 ± 2˚C, relative humidity (RH) 60 ± 10%, and photoperiod L:D = 16:8 h in laboratory conditions to acclimatize. Each pot was then covered with a 200 mesh net (40 × 30 × 100 cm), and ten M. alternatus adults were placed inside the net. The numbers of dead and surviving adults were counted daily for 25 days (25 ± 2˚C, RH 70 ± 10%, photoperiod L:D = 16:8 in laboratory conditions), and mycosis was observed. As a non-treated control, a pine tree was sprayed with 100 ml of 0.03% siloxane solution. All treatments were tested in triplicates.

Insecticidal efficacy test of ERL836 against adults using artificially larvaeinfested pine tree logs
Fifth instar M. alternatus larvae were inserted into pine logs (length 30 cm and 20 cm diameter) using 100 mm long holes that were then blocked using a piece of pine wood. Five holes were drilled per log, and one larva was placed in each hole. Then, 100 ml of B. bassiana ERL836 conidia suspension (1.0 × 10 7 conidia/ml) diluted with 0.03% siloxane solution was sprayed onto the entire log surface and dried at room temperature for 24 h. In a plastic container (30×40×30 cm 3 ), 300 g of soil was laid on the floor of the container, and 100 ml of distilled water was sprayed onto the entire soil surface. Two pine logs were placed in each plastic container, which was then sealed with a lid. The emerging adults and dead and alive adults were counted daily for 25 days (25 ± 2˚C, RH 95 ± 5%, L:D = 16:8). As a non-treated control, a pine log was treated with 0.03% siloxane solution. All treatments were tested in triplicates.
Secondly, insecticidal activity against M. alternatus was evaluated using a fungal powder formulation. The insecticidal activity of B. bassiana ERL836 against M. alternatus was evaluated in the form of a 2.5% powder formulation which was made by FarmHannong (Nonsan, Korea). The B. bassiana ERL836 powder formulation was diluted in water for spray or directly powered to pine logs. For the powder-based suspension spray, 1 and 10 g of fungal powder formulation were diluted with 100 ml water to prepare 1% and 10% suspensions, respectively. For the powdering of the formulation, 1 g and 10 g of fungal powders were scattered onto the pine logs in powder form. Plastic boxes containing pine logs artificially infected with M. alternatus larvae were prepared as described above. The entire surface of each pine log was treated with B. bassiana ERL836 according to the fungal powder treatment type. Emerging adults and dead and alive adults were counted daily for 40 days after the emergence of the first adults. All treatments were tested in triplicate in greenhouse conditions. The temperature and relative humidity were recorded every 1 hour with a HOBO U12-012 External Temperature/Relative Humidity/Light/External data logger (Onset Computer Co., Bourne, MA, USA).

Colonization of B. bassiana ERL836 on pine bark
Before we applied the B. bassiana ERL836 in the field, we assessed the ability of the fungus to colonize pine bark. Three pine barks (Pinus densiflora, P. koraiensis and P. thunbergia) were collected from trees growing wild in a forest. They were cut into discs using a cork borer (dia. 6 mm) and autoclaved at 121˚C for 15 min. A filter paper (dia. 55 mm) was placed in a Petri dish (60 × 15 mm), and a 40 × 40 mm piece of Parafilm (Heathrow Scientific, Vernon Hills, IL, USA) was placed on the filter paper to maintain high humidity. The nine pieces of bark were placed on the Parafilm at 10-mm intervals, and conidia suspensions (1.0 × 10 7 conidia/ml) of B. bassiana ERL836 and ERL836-egfp were inoculated at 20 μl per piece. The Petri dish was kept at room temperature for 24 h without a lid so that the conidia suspensions on the bark could dry naturally. Then 400 μl of distilled water was placed on the filter paper, and the Petri dish was closed and sealed with Parafilm. All treatments were maintained at 27˚C for 14 days, and then the growing fungus was observed with a microscope at × 10 magnification. The fluorescence expression of the egfp was observed with a fluorescence stereo microscope (SMZ1270, Nikon Co., Tokyo, Japan) to confirm that the growth was B. bassiana ERL836. The negative control bark was treated with 0.03% siloxane solution. All treatments were assayed in triplicates.

Semi-field test using wintering pine logs artificially infested with larvae
The semi-field test was performed from spring to fall (April to July 2020) at FarmHannong's test field (36˚07'58.1"N 127˚07'45.4"E, Nosan, Korea). Pine logs (ca. 1 m) with five final-stage larvae of M. alternatus were supplied by the insect rearing company OsangKinsect, Korea. In the test field, an iron frame (3×4×4 m) was installed, and the entire frame was covered with a black film to create shade. We treated twelve pine logs (about 60 M. alternatus larvae) with 100 g of 2.5% formulated B. bassiana ERL836 fungal powder formulation, and then the logs were piled to about 1 m 3 in the frame. The fungal-treated pine logs were covered with a covering film (tarpaulin) and sealed. Each treatment was tested in triplicate (S1A Fig). The number of emerging adults and mortality of M. alternatus adults were monitored weekly from June to July 2020. Field verified mortality in each monitoring time was pooled. Alive emerging adults were moved to a lab and kept at a moisturising Petri-dish for 14 days to assess potential infection and mortality (this data was also pooled). The temperature and relative humidity were recorded every 1 hour with a HOBO U12-012 External Temperature/Relative Humidity/Light/ External data logger (Onset Computer Co., Bourne, MA, USA). All treatments were tested in triplicates. The mortality was investigated on the day of collection and after moist treatment for 14 days after collection, and the number of alive and dead adults was calculated cumulatively for individuals collected for 6 weeks. The mortality was calculated as the ratio of the cumulative number of alive and dead adults at each time point.

Field test
The field test was performed from fall to summer (November 2019 to July 2020) in a forest area where pine wilt nematode occurred (35˚29'52.0"N 129˚17'35.8"E, Ulsan, Korea). Near that forest area, pine logs (ca. 1 m) naturally infested with M. alternatus larvae were collected with the assistance of a local forest manager during the winter (November to December 2019). The 2.5% formulation of B. bassiana ERL836 fungal powder was treated to about 50 pine logs presumed to contain about 200 M. alternatus larvae, piled to about 1 m 3 , and covered with a covering film (tarpaulin), and sealed. To evaluate the insecticidal activity according to the amount of fungal powder formulation, 100 g and 200 g of the B. bassiana ERL836 fungal powders were treated, and the non-treated control was not treated with any fungal powder. All treatments were tested in triplicate (S1B Fig). The number of emerging adults and the mortality of M. alternatus adults were monitored weekly. Field verified mortality in each monitoring time from Jun 17th to August 5th was pooled. Alive emerging adults were moved to a lab and kept at a moisturising Petri-dish for 14 days to assess potential infection and mortality, and the data was also pooled. The mortality was investigated on the day of collection and after moist treatment for 14 days after collection, and the number of alive and dead adults was calculated cumulatively for individuals collected for 8 weeks. The mortality was calculated as the ratio of the cumulative number of alive and dead adults at each time point.
To confirm the B. bassiana-mediated mortality, fungal colonies were isolated from the cadavers of M. alternatus adults and cultured in 1/4 SDA medium with 100 μg/ml of dodine (Sigma-Aldrich, Saint Louis, MO, USA), 100 μg/ml of streptomycin (Sigma-Aldrich, Saint Louis, MO, USA), and 200 μg/ml of chloramphenicol (Sigma-Aldrich, Saint Louis, MO, USA) [29]. The DNA of the fungus was extracted using a DNA extraction kit and identified by sequencing the ITS region. The full ITS region was amplified using the universal primer set ITS1F (5 0 -CTT GGT CAT TTA GAG GAA GTA A-3 0 ) and ITS4R (5 0 -TCC TCC GCT TAT TGA TAT GC-3 0 ) [30]. The temperature and relative humidity were recorded every 1 hour with a HOBO U12-012 External Temperature/Relative Humidity/Light/External data logger (Onset Computer Co., Bourne, MA, USA). All treatments were tested in triplicates.

Transmission of B. bassiana ERL836 to non-target insects
First, the direct insecticidal activity of B. bassiana ERL836 against the three non-target forest insects was investigated. The three non-target insects were A. dichotoma adult, L. maculifemoratus adult, and B. mori larvae. Each insect was placed in a plastic cup with a filter paper soaked in distilled water on the bottom of the cup, and then 1 ml of the fungal conidial suspension (1.0 × 10 7 conidia/ml) was adjusted with 0.03% siloxane solution was sprayed onto the insects. A food source was provided to each insect, and the lid of the cup was closed. The dead and alive insects were counted daily for 14 days (25 ± 2˚C). All treatments were tested in three replicates of ten insects each.
The transmission potential of B. bassiana ERL836 to the non-target forest insects was confirmed using fungus-infected M. alternatus adults. A 1-ml conidia suspension of B. bassiana ERL836 (1.0 × 10 7 conidia/ml) adjusted with 0.03% siloxane solution was sprayed onto a male adult M. alternatus and transferred to a plastic cup (lid 10 cm, bottom 60 cm, height 15 cm). The plastic cup was layered with a filter paper (dia. 55 mm), moisturized with 1 ml of distilled water. A. dichotoma adult, L. maculifemoratus adult, or B. mori larva was placed in each cup where a fungus-infected M. alternatus male adult was previously placed (one infected M. alternatus + one non-target insect per plastic cup). A pine branch (ca. 150 mm) was provided as food, and the lid was closed and sealed. The number of dead and alive adults was counted daily for 14 days (25 ± 2˚C). As a negative control, a M. alternatus male adult was treated with 0.03% siloxane solution, placed in the plastic cup with each insect, and the lid was closed. All treatments were tested in three replicates of ten adults each.

Statistical analysis
All data on the mortality of M. alternatus adults, the control efficacy, and mortality of the target and non-target insects were arc-sine transformed after an analysis of the normal distribution and then analyzed using a generalized linear model and Tukey's honestly significant difference test for multiple comparisons. All analyses were performed in SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA) at the 0.05 (α) level of significance.

Insecticidal activity of B. bassiana ERL836 against M. alternatus adults under pot conditions
In the small pot of pine tree condition, M. alternatus adults exposed to pine trees pre-treated with B. bassiana ERL836 died on nine days after the release of adults (Fig 1). The mortality of M. alternatus adults was 53.3 ± 12.0% in the 1.0 × 10 6 conidia/ml treatment and 76.7 ± 13.3% in 1.0 × 10 7 conidia/ml treatment on 25 days after release of adults. In the non-treated group, there were no dead adults on 25 days after release. Thus, the mortality of M. alternatus adults was significantly increased by the fungal pre-treatment, and it increased dose-dependently with the fungal conidia concentration (F 2,156 = 224.4, p < 0.001). In the fungal treatment groups, white conidia covered the surface of dead M. alternatus adults.

Insecticidal activity of pre-treated B. bassiana ERL836 on pine logs artificially infected with larvae
When we infested pine logs with M. alternatus larvae in indoor conditions and then treated the logs with B. bassiana ERL836, we found that M. alternatus adults began to emerge on the 30th day (Fig 2). The emergence rate of adults was counted beginning when the first adult emerged from the pine logs. The non-treated logs and B. bassiana ERL836-treated logs showed the emergence of 20.0 ± 5.8% and 23.3 ± 8.8%, respectively, on the first day and 83.3 ± 6.7% and 73.3 ± 6.7%, respectively, on the 30th day. In the non-treated group, the first adults died on 12 days after emergence, and the mortality of adults was 20.0 ± 5.8% on the 30th day. In the B. bassiana ERL836 treatment, the first adults died on the 6th day after emergence, and the mortality rate was 66.7 ± 13.3% on the 30th day. Thus, the emergence-based mortality was 88.9 ± 11.1% in the B. bassiana ERL836 treatment and 23.3 ± 5.5% in the nontreated control (Fig 2A and 2B), which was a significant difference (F 1,124 = 164.4, p < 0.001). The growth of mycelium and white conidia was observed mainly on the segment parts of the cadavers in the ERL836 treatment, which indicates that the M. alternatus adults died from exposure to the B. bassiana ERL836 (Fig 2C).
When we artificially infested pine logs with M. alternatus larvae and then treated the logs with the fungus B. bassiana ERL836 formulated as powder in greenhouse conditions, the first adults emerged from the pine logs about 90 days after the fungal treatment (Fig 3). The average temperature in the greenhouse was 16.3 ± 3.0˚C (range 8.5-28.2˚C), the relative humidity was maintained above 90%, and dark conditions were maintained. In the group treated with 1 g/ box of fungal powder, the first adults died on 12 days after their first emergence, and the emergence and mortality rates on the 30th day were 43.3 ± 3.3% and 20.0 ± 5.8%, respectively ( Fig  3A). In the 10 g/box fungal powder treatment, the first adults died on 13 days after their emergence, and the emergence and mortality rates on the 30th day were 30.0 ± 10.0% and 26.7 ± 6.7%, respectively (Fig 3B). In the 100× fungal suspension spray using the formulated powder, the first adults died on the 12th day after their emergence, and the emergence and mortality rates on the 30th day were 43.3 ± 14.5% and 30.0 ± 10.0%, respectively (Fig 3C). In the 10× fungal suspension spray using the powder, the first adults died on the 6th day after their emergence, and the emergence and mortality rates on the 30th day were 53.3 ± 8.8% and 43.3 ± 6.7%, respectively (Fig 3D). In the non-treated control, the first adults died on 17 days after their emergence, and the emergence and mortality rates on the 30th day were 23.3 ± 8.8% and 6.7 ± 3.3%, respectively (Fig 3E). There was no significant difference between the 10 g/ box powder treatment and the non-treated control (p>0.05), and the 10× suspension spray showed the highest emergence rate (F 4,310 = 36.6, p < 0.001). When we calculated the ratio of dead adults/total emerged adults on the 15th and 30th days after the first emergence of adults, the non-treated control was 0.0% and 50.0 ± 28.9%, and the other treatments were as follows (1 g/box fungal powder treatment: 25.0 ± 14.4% and 46.7 ± 14.8%, 10 g/box fungal powder treatment: 55.6 ± 29.4% and 93.3 ± 6.67%, 100× fungal suspension spray treatment: 19.4 ± 10.0% and 73.8 ± 14.5%, and 10× fungal suspension spray treatment: 68.9 ± 5.9% and 82.1 ± 9.0%, respectively) (Fig 3F). There was no significant difference between the 1 g/ box fungal powder, 100× suspension spray, and the non-treated control. The 10 g/box fungal powder and 10× suspension spray showed significantly high insecticidal activity (F 4,310 = 18.3, p < 0.001).

Fungal colonization on pine bark and M. alternatus adults
When we inoculated the sterilized bark of three pine tree species, black pine (Pinus thunbergia), red pine (P. densiflora), and white pine (P. koraiensis) with B. bassiana ERL836 and B.
bassiana ERL836-egfp, the fungus colonized on the park and conidia were produced on the bark of all three pine species (Fig 4A). To confirm that the treated B. bassiana ERL836 was growing on the bark, we inoculated B. bassiana ERL836-egfp and observed fluorescence from the fluorescent strain in the areas where the hyphae and conidia were produced. The observation of egfp fluorescence indicated that the fungus growing on the bark was B. bassiana ERL836. Thus, the B. bassiana ERL836 can colonize the bark of pine trees for mycelial growth and conidia production, suggesting that emerging M. alternatus adults from the trees could come into contact with the conidia of the fungus on the barks. On the 10th day after inoculating B. bassiana ERL836 and ERL836-egfp, mycelium and white conidia were observed in the segments of M. alternatus adults (Fig 4B). In the adults treated with B. bassiana ERL836-egfp, The 100-ml fungal conidia suspension (1.0 × 10 7 conidia/ml) was sprayed onto pine logs artificially infested with 5th instar larvae of M. alternatus, and then the logs were placed in a plastic box, and the lid was closed and sealed. Cumulative emergence of M. alternatus adults in the non-treated control (a) or ERL836 treatment (b) was counted daily after the first observation of emergence. In the emergence, the percentages of alive and dead numbers were presented as white and black, respectively. A mycotized adult was observed in the ERL836 treatment (c). https://doi.org/10.1371/journal.pone.0274086.g002

Control efficacy of B. bassiana ERL836 in semi-field and field conditions
The control efficacy from treating larvae-infested pine logs with the formulated B. bassiana ERL836 fungal powder was evaluated in semi-field conditions (Fig 5). From June 5th to July 10th, the cumulative numbers of adults that emerged from the logs of non-treated control and B. bassiana ERL836 fungal powder formulation 100 g/m 3 treatments were 32.3 ± 0.9 and 30.0 ± 0.6, respectively (Fig 5A). The weekly average numbers of adults emerging in the non-fungal powder formulation was diluted with water at 100× (c) and 10× (d) and sprayed on the logs. Non-treated control was designed (e). The cumulative emergence of M. alternatus adults was counted daily after the first observation of emergence. In the emergence, the percentages of alive and dead numbers were presented as white and black, respectively. The final mortality of each treatment was summarized (f).
https://doi.org/10.1371/journal.pone.0274086.g003 treated control and fungal powder formulation treatment were 5.4 ± 1.6 and 5.0 ± 1.4, respectively. The pooled mortalities of the adults on the investigation day in the non-treated control and fungal powder formulation were 9.0 ± 4.4% and 16.5 ± 3.5%, respectively (Fig 5B). Alive adults from the non-treated control and fungal powder treatment were collected and transferred to a lab for keeping them in a cup with high humidity for 14 days; their pooled mortalities were 40.8 ± 7.5% (non-treated control) and 87.8 ± 1.1% (ERL836 powder formulation). In the fungal powder treatment, white conidia were observed on the dead M. alternatus adults. The mortalities of adults in the non-treated control and the fungal powder treatment did not differ significantly on the investigation day, but there was a significant difference after 14 days of additional incubation under high humidity (F 3,8 = 47.5, p < 0.001).
We also tested the control efficacy of naturally infested pine logs with B. bassiana ERL836 fungal powder formulation in field conditions (Fig 6). From June 17th to August 10th, the cumulative numbers of adults that emerged in the non-treated control and B. bassiana ERL836 fungal powder 100 g/m 3 and 200 g/m 3 treatments were 126.0 ± 11.7, 90.0 ± 11.0, and 81.7 ± 19.8, respectively, and the weekly average numbers of emerged adults in each treatment group were 15.8 ± 5.6, 11.3 ± 3.9, and 10.2 ± 3.7, respectively (Fig 6A). The pooled mortalities of adults on the day of investigation in the non-treated control and B. bassiana ERL836 fungal powder 100 g/m 3 and 200 g/m 3 treatments were 4.7 ± 1.7%, 37.8 ± 2.7%, and 48.4 ± 13.0%, respectively (Fig 6B). When the adults were collected and kept under high humidity for 14 days, their pooled mortalities were 34.1 ± 4.7%, 82.1 ± 3.9%, and 87.2 ± 3.3%, respectively. In the fungal treatments, white conidia were observed on the dead M. alternatus adults. The pooled mortality of the adults on the day of the investigation was significantly higher in both fungal powder formulations of 100 g/m 3 and 200 g/m 3 (F 2,6 = 13.0, p < 0.01). The pooled mortality of the collected adults kept in the high humidity condition confirmed that all of the fungal powder formulations had significantly high insecticidal activity (F 2,6 = 39.8, p < 0.001). Our DNA sequencing results of the fungus collected from the infected M. alternatus adults and pine bark identified it as a B. bassiana isolate. Thus, B. bassiana ERL836 pre-treated on the pine bark grew and generated conidia that then killed the M. alternatus adults.

Transmission of ERL836 to non-target forest insects
The insecticidal activity of direct exposure to B. bassiana ERL836 and of M. alternatus adults treated with a B. bassiana ERL836 conidia suspension was evaluated on three species of forest insects (Fig 7). The B. bassiana ERL836-treated A. dichotoma adults had a 73.3% survival in 14 days after treatment, and the non-treated control had a 80.0% survival rate (Fig 7A). The A. dichotoma adults kept in the same cup as a fungus-treated M. alternatus adult had a survival of 83.3 ± 4.2% in 14 days contact, and the survival of the A. dichotoma adults kept in the same cup as non-treated control M. alternatus adults was 75.0 ± 5.0% (Fig 7B). The difference in the survival of A. dichotoma adults between the direct fungal exposure and infected M. alternatus  To investigate the direct insecticidal activity, ERL836 conidia exposure was not significant (F 3,156 = 271.7, p < 0.001). The survival rate of B. mori larvae treated with B. bassiana ERL836 was 0.0% in 14 days after treatment, and the survival of the non-treated control was 90.0% (Fig 7C). The survival of B. mori larvae kept in the same cup as a fungus-treated M. alternatus adult was 30.0 ± 7.3% in 14 days after exposure, whereas the survival of B. mori larvae kept in the same cup as non-treated M. alternatus adults was 55.0 ± 15.0% (Fig 7D). The difference in the survival of B. mori larvae between the two different fungal exposures did not differ significantly, and the survival of the fungus-treated M. alternatus adults did differ significantly from that of non-treated M. alternatus adults and B. mori larvae (F 3,156 = 9.8, p < 0.001). All the L. maculifemoratus adults in both the non-treated control and fungal treatment were alive in 14 days after treatment (Fig 7E), and they also survived when exposed to the fungus-treated M. alternatus adults (Fig 7F). The survival of L. maculifemoratus adults was not significantly affected by exposure to M. alternatus regardless of fungal treatment, and the survival of M. alternatus adults did differ significantly according to the fungal treatment (F 3,156 = 465.8, p < 0.001).

Discussion
In this study, we tested the potential of a commercialized entomopathogenic fungus strain, B. bassiana ERL836, as an agent for controlling the pine wilt nematode vector, M. alternatus. We devised a control strategy in which we treated the surface of pine logs where M. alternatus overwinters with the fungus instead of fumigating them with chemical agents. Then we evaluated the insecticidal activity of B. bassiana ERL836 on the emerging adults of M. alternatus. At other stages including egg, larva and pupa, M. alternatus lives inside the pine tree, which makes it difficult to evaluate the efficacy of applying fungal material to the bark. In fact, it is unreasonable to expect high insecticidal activity because this entomopathogenic fungi do not penetrate the bark except some of endophytic isolates; the insects make a tunnel inside the pine tree during the winter and form a chamber around their bodies for protection [31]. When the adults leave the overwintering pine tree logs to feed on fresh leaves, they make contact with the pine bark and thus are exposed to the fungus at that time. That is the starting point of the insecticidal mechanism of the fungus against M. alternatus because after an adult emerges from the log, it feeds at the bark, allowing fungal contact to be maintained continuously and produce infection. To use the life cycle of M. alternatus as a control mechanism, it is essential for the fungus to colonize the pine bark and maintain its activity over the winter. To test whether the fungus could be used in the field, we evaluated its insecticidal activity against M. alternatus when the fungus was treated to the pine bark before emergence, its colonization on the surface of the pine bark, and the control efficacy of our formulated fungal powder in semi-field and field conditions. In addition, we considered the stability of forest biota by evaluating the possibility that infected M. alternatus could transmit the fungus to non-target forest insects. The results of this study demonstrate that B. bassiana ERL836 can be used as an environment-friendly agent to effectively control the pine wilt vector M. alternatus.
M. alternatus feeding on pines pre-treated with the fungus had a mortality of 70% in 25 days after treatment at 1.0 × 10 7 conidia/ml, and the insecticidal activity was delayed. The high humidity condition was not maintained in this experiment because the pine growing small pots were not sealed; there was only an installed 200 mesh net only to prevent the M. alternatus adults from escaping from the individual pot. However, we did confirm that M. alternatus could be killed by the fungus remaining on the pine tree, which can be expected to have a long-term insecticidal activity against pests that attack the tree. We found that the higher the concentration of conidia, the higher the insecticidal activity, so if fungal conidia were treated to living pine trees, the insecticidal activity could be expected to be dose-dependent. In other words, even if the fungal conidia are not treated directly to M. alternatus, the fungi remaining in the pine trees could have insecticidal effects on pests. However, such a treatment method would require insecticidal tests against non-target insects because fungi can affect other nearby insects when it is treated to pine trees. The application of fungal conidia in forests could adversely affect the ecosystem if it affected non-target insects, so it is difficult to apply in forests below the economic control level, except in areas where PWN is a serious problem. Among the methods for using entomopathogenic fungi to control M. alternatus, we focused on strategies that treat the fungus to piled pine logs already known to be infected with PWN and insect vectors. Using the management strategies we tested, even a high concentration of insecticide sprayed in a sealed state within a certain area has relatively few adverse effects on the surrounding environment, and high control efficacy can be expected. Previously, we conducted a control study by applying the conidia of a M. anisopliae isolate on piled wintering pine logs [20]. We confirmed that the fungus colonized the pine tree bark, which suggested the possibility of controlling M. alternatus by treating overwintering logs with the fungus.
When M. alternatus adults emerged from inside a tree treated with the B. bassiana ERL836 conidia suspension, they ate the fungal-treated pine bark, which put them into continuous contact with the fungus. In field conditions, we predicted that the frequency of M. alternatus adults piercing the covering film and escaping into the surrounding forest would be small. In addition, the conditions for sealing larvae-infested pine logs with a covering film can maintain the relative humidity inside in a saturated state, thereby providing conditions favorable to the growth of fungi. B. bassiana ERL836 had high insecticidal activity against M. alternatus adults in the sealed condition, which demonstrates that the activity of the fungus and its insecticidal activity are maintained in high humidity.
B. bassiana ERL836 was observed to grow on the bark of pine trees (P. koraiensis, P. densiflora, and P. thunbergia) and produced fungal conidia. We observed that mycelium and conidia were generated on the bark of trees treated with the B. bassiana ERL836-egfp strain, and fluorescence was observed in all the places the fungus grew. On the bark treated with B. bassiana ERL836, the fungus grew and produced conidia, but no fluorescence was observed, and no fluorescence was observed in the non-treatment group. Therefore, B. bassiana ERL836 grew on the bark, as suggested not only by the fungal conidia on the treated surface of the pine bark but also by the fungi that colonized the bark and produced additional conidia, which is expected to positively affect the insecticidal activity for a long time.
B. bassiana ERL836 is a commercialized strain already being used to control thrips. In this study, this strain had high insecticidal activity against M. alternatus. This commercialized strain can be applied in various forms depending on the stability and use of the fungus. In this study, we devised an application method to replace chemical fumigants by applying the fungus to overwintering pine tree logs known to be infested by M. alternatus. We evaluated our method in a greenhouse, semi-field, and field conditions: we treated our powder formulation itself by powdering and diluted the powder in water for spray. In the greenhouse conditions, all treatments showed a low emergence rate of M. alternatus adults, which might have been due to the relatively low temperature and daily temperature fluctuations in the greenhouse (8.5-28.2˚C, average 16.3˚C.). However, the insecticidal activity of the fungus was maintained, and dead M. alternatus were observed in the fungal treatment groups. After the fungal suspension spray, the adults died 6-11 days after emergence, and on the 30th day, the percentages of dead individuals among the emerging adults were 73.8% and 82.1% with the 100× and 10× fungal suspension sprays, respectively. On the other hand, after the fungal powder treatment, the adults died 11-12 days after emergence, and on the 30th day, the percentages of dead individuals among the emerging adults were 46.% and 93.3% with the 1 g/box and 10 g/box fungal powder treatments, respectively. Based on the 1 g fungal treatments (both 1 g/box powder and 100× suspension spray using 1 g), the fungal suspension spray had higher insecticidal activity than the powdering, probably because the fungal conidia diluted and suspended in water were treated and colonized the bark evenly, which gave a high probability that the emerging adults would contact the fungal conidia. On the other hand, the powdering showed relatively low insecticidal activity because it could not completely cover the trees even if it was treated evenly across the bark.
However, the 10 g/box powderings covered a larger area than the 1 g/box powdering, increasing both the chance that an adult would contact it and the amount of fungal conidia that attached to the adults and producing insecticidal activity similar to that of the 100× fungal suspension spray. Therefore, control efficacy using the powder form requires enough fungal powder to adequately cover the pine logs within the treated compartment. Thanks to the sealed characteristics of our control strategy, it does not have a significant adverse effect on the environment, even if the fungus is treated at a high concentration. Therefore, the fungal powdering, which is more convenient to use than the suspension spray, seemed suitable for controlling M. alternatus in a big forest situation. To test that possibility, we evaluated the control efficacy against M. alternatus in semi-field and field conditions using the formulated fungal powder with the powdering application method. The suspension spray method has a fatal weakness in that it requires water to be moved to forest areas. Thus, a vehicle is required to move a large amount of water to a forested area, making it difficult to apply a suspension in an area where suitable transportation is unavailable. On the other hand, the fungal powder method for treating M. alternatus larvae-infested pine is a simple matter of carrying the needed amount of powder and then scattering it.
The environmental conditions in which the fungus is applied require a formulation stable in both winter and summer. In fungal biopesticides, the temperature is an important factor. The fungus needs to survive on the pine bark during the low temperatures of winter, and needs to be active when the adults emerge from the pine tree in the spring. The B. bassiana ERL836 was able to maintain conidia activity for more than two years at a temperature of 4-30˚C in previous studies [32]. In addition, it has been reported that B. bassiana isolates survive at even the low temperature of -15˚C and continue to have insecticidal activity when the temperature becomes suitable for growth [33]. The average winter temperature from 2001 to 2020 in Korea was -1.0~3.1˚C, with a minimum of -12.6˚C (Statistics Korea, 2021), so fungal conidia can survive the winter. An additional effect of protecting fungal conidia against cryogenic temperatures can be expected by covering the fungal-treated pine logs with the covering film, tarpaulin. The conidia activity of B. bassiana ERL836 can thus be maintained until the spring of the following year, when the M. alternatus adults emerge, come into contact with it, are infected, and die. The ERL836 can maintain long-term activity at a temperature of up to 30˚C, maintain a conidial germination rate of more than 80% even when stored at 30˚C for 18 months, and maintain a high level of insecticidal activity on insects. In other B. bassiana strains, fungal activity was maintained up to 34˚C, but at temperatures higher than that, the activity decreased sharply, making insecticidal activity unlikely [18,32]. Maintaining insecticidal activity for two years is an important factor in controlling M. alternatus because the wintering period varies depending on the stage of the larvae. During the wintering period, the larvae of the fourth and fifth instars emerge as adults the following year, but the first-third instars do not emerge until the spring after two winters [34]. In other words, to completely control M. alternatus, the insecticidal activity of the fungus must be maintained for close to two years. The B. bassiana ERL836 can be maintained for a long time, so it has a great advantage in controlling M. alternatus. However, because large temperature changes can greatly reduce the stability of fungi, the stability of B. bassiana ERL836 should be evaluated under alternating temperature conditions. Forest environments are more diverse in their biota than agricultural environments. It is difficult to selectively show non-pathogenicity to insects due to the characteristics of fungi, so it is important to seal fungal treatments in specific areas when using them in forest environments. However, M. alternatus adults could plausibly escape the covering film securing the fungustreated pine log pile, so whether fungus-infected M. alternatus adults can transmit the fungus to non-target insects is a major factor in its suitability for use as a biological pesticide in forests. In our results, ERL836 had low virulence in Japanese rhinoceros beetle (A. dichotoma) adults and Korean stag beetle (L. maculifemoratus) adults, but high virulence in silkworm (B. mori) larvae. The results of directly spraying a high concentration of the fungus indicated low virulence against two coleopteran forest insects except silkworm, and the transmission of fungus by fungus-treated adult M. alternatus was deemed unlikely in those two species. In the case of the silkworm larvae, the survival rate was low even in the non-treated control group when kept with non-infecting M. alternatus adults, so other factors, such as stress, are the likely cause, rather than the fungus. In the evaluation of transmission potential, the M. alternatus adults also had a low survival rate, even in the non-treated control group, so stress or fighting with other insects in a confined space is deemed to be the cause of death rather than fungal infection. In a previous experiment, we observed that when adults of the same species were placed in a small space, antennae and other body parts were damaged by fighting with each other (data not shown). The results of the transmission evaluation thus indicate that the impact on the environment from this control strategy is low because ERL836 has relatively low virulence against non-target insects in the forest although the tested insect number was small, and the infected M. alternatus adults rarely transmit the fungus to other species of insects in close quarters, and the possibility of contact with one another in forest conditions is low due to the covering film.
Finally, we proposed a strategy to control M. alternatus by pre-treating the overwintering larvae-infested pine logs with the fungus B. bassiana ERL836 (Fig 8). That strategy can be easily carried out using the formulated fungal powder, and the treated fungus can colonize the pine bark and have insecticidal activity limited to M. alternatus within the treatment area. In addition, because the control strategy calls for sealing the treated logs, it has the advantage of being able to ensure high humidity and temperature, which maximizes both fungal activity and its expected insecticidal activity against adult insects. We confirmed that the possibility of killing other insects is low even if an infected M. alternatus adult is exposed to the forest, and we verified that the fungus can be used as a high-efficacy and eco-friendly control agent in forests. However, we can consider one more thing to use entomopathogenic fungi as biopesticides. Although it has been proven that the control efficacy against pest using entomopathogenic fungi through this strategy is high, economic feasibility must be obtained to use formulated entomopathogenic fungi. Fortunately, the entomopathogenic fungus B. bassiana ERL836 used in this study are already commercialized strains, and it is proved that they have been completed to a considerable degree in culture and formulation. However, in terms of control efficacy and application, it is necessary to compare with chemical agents used previously, and by comparing them, it is proved that the economic value of control using biological pesticides can be improved.
Pine wilt disease, which is caused by pine wilt nematodes, is a major problem in Asia, Europe, and other regions. Eco-friendly materials are needed to replace the chemical fumigation agents currently used to control overwintering insect vectors that mediate pine wilt nematodes. In this study, we used an entomopathogenic fungus, Beauveria bassiana ERL836, to replace the previously used chemical fumigant-based control method on pine trees where Monochamus alternatus had been found during overwintering. B. bassiana ERL836 had great insecticidal activity against M. alternatus adults, and excellent colonization on pine tree surfaces by generating mycelium and conidia. The fungus can withstand low winter temperatures on pine tree bark and act as a deadly pathogen to M. alternatus adults that emerge from the tree logs after overwintering. A formulated B. bassiana ERL836 fungal powder treatment on the tree log surfaces is an effective and efficient control strategy, although both of powder and powder-based suspension treatments showed similar insecticidal activity. Rather than diluting the fungal powder in water for sprayable suspension, fungal powder applications do not require equipment beyond a container in which to keep the preparation. Treating the fungal powder to piled and damaged pine tree logs had a low probability of transmitting the fungus to other forest insects, which is an important advantage; entomopathogenic fungi can preserve forest biodiversity except for the target pest. Therefore, the fungal powder preparation has high value as an eco-friendly control strategy that can effectively protect the forest environment. The results presented here could make a great contribution to the eco-friendly management of pine trees by enabling the application of entomopathogenic fungi to major pine forests to control pine wilt nematode vectors.

Conclusions
Pine wilt disease, which is caused by pine wilt nematodes, is a major problem in Asia, Europe, and other regions. Eco-friendly materials are needed to replace the chemical fumigation agents currently used to control overwintering insect vectors that mediate pine wilt nematodes. In this study, we used an entomopathogenic fungus, Beauveria bassiana ERL836, to replace the previously used chemical fumigant-based control method on pine trees where Monochamus alternatus had been found during overwintering. B. bassiana ERL836 had great insecticidal activity against M. alternatus adults, and excellent colonization on pine tree surfaces by generating mycelium and conidia. The fungus can withstand low winter temperatures on pine tree bark and act as a deadly pathogen to M. alternatus adults that emerge from the tree logs after overwintering. A formulated B. bassiana ERL836 fungal powder treatment on the tree log surfaces is an effective and efficient control strategy, although both powder and powder-based suspension treatments showed similar insecticidal activity. Rather than diluting the fungal powder in water for sprayable suspension, fungal powder applications do not require equipment beyond a container in which to keep the preparation. Treating the fungal powder to piled and damaged pine tree logs had a low probability of transmitting the fungus to other forest insects, which is an important advantage; entomopathogenic fungi can preserve forest biodiversity except for the target pest. Therefore, the fungal powder preparation has high value as an eco-friendly control strategy that can effectively protect the forest environment. The results presented here could make a great contribution to the eco-friendly management of pine trees by enabling the application of entomopathogenic fungi to major pine forests to control pine wilt nematode vectors.